Myosin X was first identified by PCR using primers to two conserved regions of the myosin heavy chain from a frog inner ear library. Clones corresponding to the entire sequence were subsequently obtained from a bovine smooth muscle library. The sequence revealed a conserved motor domain followed by three IQ motifs that are potential light chain binding domains. There is a short segment predicted to form a coiled- coil that probably allows for dimerization to produce a two-headed molecule. Virtually nothing is known about myosin Xs function or localization within cells. In order to characterize the in vitro enzymatic properties of this myosin, we have engineered a 961 amino acid fragment corresponding to an HMM-like fragment of bovine myosin X for expression in Sf9 cells. This fragment includes the entire motor and neck region and the sequence predicted to form a coiled-coil region. A FLAG-epitope was engineered onto the carboxyl terminus to facilitate purification. We first co-infected Sf9 cells with the myosin X heavy chain-containing virus along with a virus that contained calmodulin. Soluble myosin containing bound calmodulin was obtained by FLAG-affinity chromatography. The myosin X-HMM-like fragment binds actin in an ATP-dependent manner and has an actin-activated MgATPase with a Vmax of 10 per sec and a Km of 5 micromolar at 37C. The MgATPase is relatively ionic strength insensitive compared to conventional myosin IIs. The myosin X-LMM translocates actin filaments in a rate of 0.18+0.05 microns/s in the in vitro motility assay. Motility requires moderate densities of myosin bound to the surface and is enhanced by the presence of methylcellulose, a viscosity inducing reagent. Thus, myosin X does not appear to be a candidate for a processive vesicle transporter. We are engineering a S1-like fragment of the myosin X for further kinetic studies. - Myosin X;unconventional myosin;baculovirus;in vitro motility